Theta (?)-defensins are 18-amino acid macrocyclic peptides expressed in leukocytes of Old World monkeys(OWMs) and are the only examples of backbone-cyclized polypeptides in animals. Sixteen ?-defensin isoforms, all sharing an invariant 10-amino acid core structure, are expressed in neutrophils of OWMs. ?-defensin isoforms block the expression of TNF in stimulated leukocytes with IC50's varying from 40-350 nM. ?-defensin-mediated TNF blockade is rapid and results in the down regulation of other inflammatory cytokines in vitro and in vivo. Multiple lines of evidence indicate that one mechanism mediating the anti-TNF effect is the competitive inhibition of TNF-? converting enzyme (TACE; ADAM17), suggesting that ?-defensins are endogenous regulators of TACE. ?-defensin isoforms also inhibit the activities of other key inflammatory proteases of the matrix metalloprotease and cathepsin families. RTD-1, the prototype ?-defensin, arrests rat pristane-induced arthritis (PIA), a model of rheumatoid arthritis (RA). The overall goal of this project is to further characterize the cellular and molecular mechanisms that mediate the anti-inflammatory properties of ?-defensins. Three Specific Aims are proposed, each of which exploit the natural structure-function diversity of ?-defensins: In Aim 1: in vitro cellular assays will be used to identify inflammatory cascades that are modulated by ?-defensins; the role of TNF/TACE-blockade will be analyzed by RNA inhibition of TACE in THP- 1 cells; paracine/autocrine effects of ?-defensins will be analyzed by quantifying peptide biosynthesis/secretion by stimulated macaque granulocytes and determining the immunomodulatory effects of releasates on resting and activated target cells; analogous studies will be conducted with RTD-1-expressing THP-1 transfectants to evaluate the immunoregulatory and antimicrobial properties conferred to transfected cells. Studies in Aim 2 will utilize novel in silico docking protocols, biochemical and structure-activity analysis, and analog design to analyze the interactions of ?-defensins with TACE and other proinflammatory proteases that are inhibited by ?-defensin isoforms and which are implicated in RA pathogenesis. In Aim 3, the rat PIA model will be used to analyze the effects of systemically administered ?-defensins. Using this model, three ?-defensin isoforms, predicted (based on results of Aims 1 and 2) to have different levels of efficacy, will be evaluated pharmacokinetically and pharmacodynamically: therapeutic response, peptide levels in serum and joint tissue, serum and joint biomarkers, and histology of joint tissues will be analyzed over the course of disease progression and resolution. Collectively, these studies will provide new and significant insights into the regulation of systemic inflammation as revealed by the mechanisms mediated by endogenous immunoregulatory peptides of OWM, and will disclose the potential for 'resurrecting' ?-defensins as therapeutics for human inflammatory disorders.